scholarly journals Inhibition of DNA synthesis in adrenocortical cells by cytochalasin B.

1980 ◽  
Vol 86 (1) ◽  
pp. 129-134 ◽  
Author(s):  
M A McPherson ◽  
J Ramachandran
Keyword(s):  
Dna Synthesis ◽  
Cytochalasin B ◽  
Thymidine Incorporation ◽  
Cell Types ◽  
Mouse Tumor ◽  
Adrenocortical Cells ◽  
Normal Cells ◽  
Normal Rat ◽  
Thymidine Transport ◽  
Inhibition Of Dna Synthesis

ACTH inhibits DNA synthesis in normal rat and mouse tumor Y-1 adrenocortical cells within the same concentration range that it stimulates steroidogenesis. These processes can be independently regulated as demonstrated by the divergent actions of cytochalasin B on these cells. In the normal cells, cytochalasin B does not increase steroidogenesis in serum-free or serum-containing media, and it decreases the stimulation produced by ACTH. In the absence of serum, the Y-1 cells respond in a similar way. However, in serum-containing media, cytochalasin B increases steroidogenesis in these cells and does not inhibit the response to ACTH. In both cell types, cytochalasin B inhibits [3H]thymidine incorporation into DNA by a mechanism different than that of ACTH. In the Y-1 cells, this inhibition is caused by a decreased uptake of [3H]thymidine into the cell, which probably reflects a decreased transport across the cell membrane. In the normal cells, cytochalasin B, like ACTH, does not affect [3H]thymidine transport, but it decreases DNA synthesis much more rapidly than does ACTH. This inhibition may be the result of the disruption of microfilaments by cytochalasinB, because our evidence indicates that it is not caused by a decrease in glucose uptake by the cells.

Effect of cytochalasin B on somatic cell hybrids between normal and transformed cells

1985 ◽  
Vol 78 (1) ◽  
pp. 87-96
Author(s):  
I. Hickey ◽  
C. McConville ◽  
M. McMenamin ◽  
R. Neill
Keyword(s):  
Dna Synthesis ◽  
Somatic Cell ◽  
Cytochalasin B ◽  
Nuclear Division ◽  
Transformed Cells ◽  
Somatic Cell Hybrids ◽  
Recessive Character ◽  
Normal Cells ◽  
Cell Hybrids ◽  
Transformed Cell Lines

Cytochalasin B (CB) prevents cytokinesis in animal cells. In normal cells nuclear division and DNA synthesis are also blocked and the cells, held in the G1 phase of the cell cycle, remain either mononucleate or binucleate. In transformed cell lines DNA synthesis and nuclear division continue and the cells become multinucleate. We have examined the response to CB in two sets of somatic cell hybrids made between cells that display multinucleation after CB treatment and cells that do not. In a cross between transformed mouse LMTK cells and normal rat embryo lung cells, very little multinucleation was observed after treatment with CB for 7 days. The ability of the LMTK cells to form clones in soft agar was also significantly reduced in these hybrids. Segregant sub-clones that re-expressed both of these transformation phenotypes were isolated. These had reduced chromosome numbers. A second cross was made between two variants of the BHK cell line, one of which displayed a high level of multinucleation in CB while the other did not. Again the hybrids showed a response similar to that of the non-multinucleating parent. From the results obtained with these two hybrids we conclude that the multinucleation induced in transformed cells by CB behaves as a recessive character in crosses with normal cells.

Cell site and time course of DNA synthesis in pancreas after caerulein and secretin

1983 ◽  
Vol 245 (1) ◽  
pp. G99-G105 ◽  
Author(s):  
T. E. Solomon ◽  
M. Vanier ◽  
J. Morisset
Keyword(s):  
Dna Synthesis ◽  
Time Course ◽  
Thymidine Incorporation ◽  
Cell Types ◽  
Cell Labeling ◽  
Dna Labeling ◽  
Dna And Rna ◽  
Rna Content ◽  
Total Dna ◽  
Two Populations

Pancreatic weight, [3H]-thymidine incorporation into DNA, labeling indices, and total DNA and RNA content were measured in rats treated with vehicle or 1 microgram/kg caerulein, 100 micrograms/kg secretin, or a combination of these peptides injected every 8 h for 1-5 days. Incorporation of [3H]thymidine into DNA increased 12-fold after 2 days of treatment with the combination of peptides. DNA content increased after 3 days and reached a level 1.8 times control after 5 days. Autoradiography showed that two cell types, acinar and an unidentified type, were the sites of increased DNA synthesis. Different patterns of labeling were seen in the two populations: acinar cell labeling indices were increased at 1 and 2 days (20-fold) and then fell; nonacinar cells showed an increase only after 2 days and maintained this increase after 5 days. Potentiation (greater than additive effects) was found when caerulein and secretin were injected together for all measurements except RNA content. These data indicate that DNA synthesis in two cell populations is affected by secretin and caerulein and support the occurrence of potentiation between secretin and caerulein for trophic effects on the exocrine pancreas.

Influence of food deprivation and adrenal steroids on DNA synthesis in various mammalian tissues

1975 ◽  
Vol 228 (1) ◽  
pp. 310-317 ◽  
Author(s):  
AL Goldberg ◽  
DF Goldspink
Keyword(s):  
Dna Synthesis ◽  
Food Deprivation ◽  
Thymidine Incorporation ◽  
Normal Growth ◽  
Adrenal Steroids ◽  
Muscle Weight ◽  
Mammalian Tissues ◽  
Influence Of Food ◽  
Inhibition Of Dna Synthesis ◽  
Fasted Rats

The DNA content of skeletal muscle increases as young rats grow. Food deprivation prevented this increase: total DNA remained constant, while muscle weight and RNA decreased. Diaphragms isolated from fasted rats incorporated [3H]thymidine into DNA far more slowly than tissues from fed rats. Incorporation returned to control levels on refeeding. Fasting for 24 or 48 h also markedly reduced [3H]thymidine incorporation by slices of liver, kidney, and brain. The factors responsible for this inhibition of DNA synthesis were investigated. Amino acids, insulin, or serum from fed or fasted rats failed to alter thymidine incorporation by muscle. Injection of hydrocortisone into normal rats reduced incorporation into kidneys, liver, and muscle within 4h. Incubation of hemidiaphragms with hydrocortisone suppressed [3H]thymidine incorporation within 2-3h. Adrenalectomy enhanced incorporation into DNA by diaphragm, liver, kidney, and brain. When fasted, adrenalectomized rats showed little or no suppression of [3H]thymidine incorporation and lost less weight than fasted controls. These data suggest that adrenal steroids are important in inhibiting DNA synthesis during normal growth and during fasting.

scholarly journals THYMIDINE TRANSPORT BY CULTURED NOVIKOFF HEPATOMA CELLS AND UPTAKE BY SIMPLE DIFFUSION AND RELATIONSHIP TO INCORPORATION INTO DEOXYRIBONUCLEIC ACID

1972 ◽  
Vol 55 (1) ◽  
pp. 161-178 ◽  
Author(s):  
Peter G. W. Plagemann ◽  
John Erbe
Keyword(s):  
Thymidine Incorporation ◽  
Initial Rate ◽  
Hepatoma Cells ◽  
Novikoff Hepatoma ◽  
Simple Diffusion ◽  
Transport Reaction ◽  
Rate Limiting Step ◽  
Low Concentrations ◽  
Thymidine Transport ◽  
Inhibition Of Dna Synthesis

The initial rate of thymidine-3H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 µM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22°, 27°, 32°, and 37°C with an apparent Km of 0.5 µM, and the Vmax values increased with an average Q10 of 1.8 with an increase in temperature. The intracellular acid-soluble 3H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 µM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 µM p-chloromercuribenzoate for 15 min or heat-shock (49.5°C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 µM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent Km and Ki values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 µM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.

scholarly journals cAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

2005 ◽  
Vol 16 (3) ◽  
pp. 1527-1542 ◽  
Author(s):  
Soheil Naderi ◽  
Jean Y.J. Wang ◽  
Tung-Ti Chen ◽  
Kristine B. Gutzkow ◽  
Heidi K. Blomhoff
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Antitumor Agents ◽  
Cell Types ◽  
S Phase ◽  
3T3 Fibroblasts ◽  
Phase Progression ◽  
Inhibition Of Dna Synthesis

cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1proteins, since both processes were only marginally affected by increased levels of cAMP in Rb-/-and p21Cip1-/-3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase–specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase–specific antitumor agents.

Effects of X-radiation on thymidine incorporation and deoxyribonucleoprotein integrity in rat tissues

1969 ◽  
Vol 47 (11) ◽  
pp. 1003-1006 ◽  
Author(s):  
D. K. Myers ◽  
S. Ram
Keyword(s):  
Dna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Thymidine Incorporation ◽  
Rat Tissues ◽  
Lethal Effects ◽  
Early Effect ◽  
Inhibition Of Dna Synthesis ◽  
Postradiation Period ◽  
Observation Period

The early effect of X-radiation on thymidine incorporation into deoxyribonucleic acid (DNA) did not differ markedly in rat tissues which are resistant and those which are sensitive to the lethal effects of irradiation. Inhibition of DNA synthesis was accompanied by loss of integrity of deoxyribonucleoprotein (DNAP) in all tissues examined during the postradiation period. The cells of radioresistant tissues were able to repair the postradiation damage to DNAP, while the degradation of DNAP continued unchecked in radiosensitive tissues during a 24-h observation period.

scholarly journals Altered protein secretion and extracellular matrix deposition is associated with the proliferative phenotype induced by allylamine in aortic smooth muscle cells

1993 ◽  
Vol 289 (1) ◽  
pp. 57-63 ◽  
Author(s):  
K S Ramos ◽  
T J Weber ◽  
G Liau
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Dna Synthesis ◽  
Protein Secretion ◽  
Thymidine Incorporation ◽  
Cell Types ◽  
Proliferative Potential ◽  
Matrix Deposition ◽  
Altered Protein ◽  
Extracellular Matrix Deposition

Repeated cycles of allylamine-induced aortic injury in vivo modulate the proliferative potential of smooth muscle cells (SMCs) during serial propagation in vitro. This modulation may be partly mediated by disturbances in polyphosphoinositide metabolism which afford allylamine-treated cells a growth advantage over control cells [Cox, Murphy and Ramos (1990) Exp. Mol. Pathol. 53, 52-63]. The present studies were conducted to further evaluate the mechanisms which mediate the enhanced proliferative potential of allylamine cells. Cellular growth and/or [3H]thymidine incorporation into DNA were evaluated in control and allylamine cells seeded on plastic culture dishes or glass coverslips in the presence of 0.1, 1 or 10% fetal bovine serum (FBS). On either substrate, incubation in 0.1% FBS for 48 h inhibited DNA synthesis in cultures of both cell types, but the inhibitory response was more pronounced in allylamine cells. Subsequent challenge with 10% FBS increased thymidine incorporation to a greater extent in allylamine cells. Interestingly, enhanced DNA synthesis of allylamine cells was associated with increased cell numbers only when seeded on a glass surface. The enhanced growth rate on glass was not due to increased plating efficiency since comparable attachment rates were observed for both cell types. Reseeding of control cells on glass substrates pre-coated by allylamine cells afforded control cells a growth advantage comparable with that observed for allylamine cultures. Conditioned media from growth-arrested, as well as cycling cultures, of allylamine cells stimulated DNA synthesis in cultures of either cell type to a greater extent than conditioned media from control counterparts. In addition, the responsiveness of allylamine cells to secreted products was enhanced relative to that of control cells. Metabolic labelling studies revealed that the synthesis and/or secretion of 52, 46, 33 and 28 kDa proteins was enhanced in allylamine cultures relative to controls, and that the expression of two proteins of 30 and 31 kDa only occurred in allylamine cultures. We conclude that the enhanced growth response of allylamine cells is associated with both altered protein secretion and differential extracellular matrix deposition.

Nitric oxide and superoxide in cultured cells: limited production and influence on DNA synthesis

1993 ◽  
Vol 265 (3) ◽  
pp. R518-R523
Author(s):  
C. Firnhaber ◽  
M. E. Murphy
Keyword(s):  
Dna Synthesis ◽  
Cultured Cells ◽  
Thymidine Incorporation ◽  
Mesangial Cells ◽  
Cell Types ◽  
Skin Fibroblasts ◽  
Lung Fibroblasts ◽  
Embryonic Lung ◽  
Opposing Effects ◽  
Lines Of Evidence

The ability of superoxide (O2-) and NO to influence DNA synthesis, as estimated by [3H]thymidine incorporation in logarithmically growing cells, was evaluated in three human cell types: embryonic lung fibroblasts, skin fibroblasts, and mesangial cells. Nontoxic rates of O2- generation (0.2 nM/min-0.2 microM/min) only slightly stimulated DNA synthesis in mesangial cells (10-13% increase) and did not affect fibroblast DNA synthesis. Nontoxic rates of NO generation (0.1-10 microM/min) also had only limited effects, inhibiting DNA synthesis in lung and skin fibroblasts slightly (10-20% decrease) but not affecting mesangial cells. In all three cell types, neither O2- nor NO was generated at high enough endogenous rates to support an autocrinic regulation of DNA synthesis. This and other lines of evidence indicate that a reaction between O2- and NO is unlikely to account for their opposing effects on DNA synthesis.

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